Preparation and use of cyclic and branched peptides and their labelled derivatives as therapeutic agents, cholecystokinin agonists or antagonists, and diagnostic agents to identify and locate tumours

ABSTRACT

This patent application describes the preparation of cyclic and branched peptides of general formula (I) and their conjugated derivatives labelled with a paramagnetic or radioactive metal. The compounds of the present invention are used as diagnostic agents to identify and locate primary tumours and their metastases which over-express type A and/or B cholecystokinin receptors, and as therapeutic agents and cholecystokinin agonists or antagonists.

The present invention relates to cyclic and branched peptides of generalformula (I) and their derivatives conjugated with a spacer molecule Yand a chelating agent C, labelled with a paramagnetic or radioactivemetal.

The compounds of the invention are used as diagnostic agents to identifyand locate primary human tumours and their metastases which over-expresstype A and/or type B cholecystokinin receptors, and as therapeuticagents and cholecystokinin agonists or antagonists.

Cholecystokinins (CCKs) are a family of peptide molecules whosebiological action is performed as a hormone and a neurotransmitter. Allthe CCKs originate from a process of fragmentation which takes place ona pre-hormone consisting of 115 amino acid residues, followed by apost-translational process of alpha-amidation of the C-terminalphenylalanine residue and sometimes, sulphation of the tyrosine residuecontained in the C-terminal portion. The cholecystokinins thereforeexist in various molecular forms; the most important ones have asequence of 58, 39, 33 or 8 amino acid residues, and they all have thesame C-terminal sequence of 8 amino acid residues:

-   -   Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-amide.

The form containing this sequence only is known as CCK8.

The biological activity of cholecystokinin depends on the type ofreceptor with which it interacts. Two types of receptor are known: typeA and type B. In non-pathological situations, type A receptor is presentin the tissues of peripheral organs such as the stomach, gall bladder,intestine and pancreas. The most important physiological actions due tothe interaction of the CCK peptide hormone with type A receptor arecontraction of the gall bladder, secretion of pancreatic enzymes,regulation of secretion, and absorption into the gastrointestinal tract.Type B receptor is mainly present in the central nervous system, wherethe interaction with cholecystokinin causes analgesia, satiety andanxiety, and regulates the release of dopamine.

Both the cholecystokinin receptors belong to the class of G-ProteinCoupled Receptors (GPCRs), membrane receptors with seven transmembranehelixes joined by intra- and extra-cellular loops with an extracellularN-terminal arm and an intracellular C-terminal part. Both receptors havehigh affinities for the various forms of cholecystokinin; however, typeA receptor has a greater affinity for the sulphated forms ofcholecystokinin, namely the ones which contain a sulphuric group on theTyr 27 residue, while type B receptor has a high affinity for thevarious forms of non-sulphated cholecystokinin and for gastrin. A seriesof peptide and non-peptide cholecystokinin-analog molecules with agonistor antagonist activity for type A and type B receptors are known (P. DeTullio, Current Medicinal Chemistry, 6, 433, 1999; F. Noble, Progress inNeurobiology, 58, 349, 1999). No pharmacological application has beenfound for any of the known molecules due to their low bioavailabilityand low solubility or high enzymatic degradation.

Cholecystokinin receptors have recently been identified in primary humantumours and metastases (J. C. Reubi, Cancer Research, 57, 1377, 1997,WO9731657). The use of functional peptides labelled with radioactivemetals such as ¹²⁵I (Biochemical Journal, 89, 114-123, 1963), ¹¹¹In or¹¹⁵In, used in nuclear medicine to visualise human tumours, is describedin particular in that article and in the patent cited by J. C. Reubi.

Type A receptor in particular is over-expressed in pancreatic andoesophageal tumours, while type B receptor has been found to beover-expressed in small lung cell tumour, tumours of the colon andgastrointestinal tract, medullary thyroid tumours, astrocytomas andovarian stromal tumours.

Some peptides deriving from cholecystokinin modified with chelatingagents of radioactive or paramagnetic metals have been studied inclinical trials. In particular, CCK8 derivatives containing thechelating agents DTPA or DOTA which complex radioactive metals like¹¹¹In and ⁹⁰Y, and their application to identify and treat tumours thatover-express type B cholecystokinin receptor, have been reported (M. DeJong, Journal of Nuclear Medicine, 40, 2082, 1999).

The NMR structure of the complex between the non-sulphated peptide CCK8and the N-terminal part of type A cholecystokinin receptor, responsiblefor the interaction with the peptide hormone, was recently published (M.Pellegrini, Biochemistry, 38, 14775, 1999). The N-terminal part of thereceptor (receptor fragment) consists of 47 amino acids, and representsthe extracellular N-terminal arm and the first part of transmembranehelix 1 of the type A receptor. This fragment does not contain theresidue of Arg 197, present on the transmembrane loop, which isresponsible for the interaction with the sulphuric group of Tyr 27 ofCCK 8, with the result that peptide CCK8 is not used in the sulphatedform (V. Gigoux, Protein Science, 8, 2347, 1999). In addition to thedetailed structural information indicated in the NMR study, a recentstudy performed by observing the variations in fluorescence of thetryptophan residues present on the receptor fragment and the peptideconfirmed the binding (R. Ragone, Biopolymers, 47-53, 56, 2001,publication pending), and enabled the affinity constant betweennon-sulphated CCK8 and the receptor fragment to be determined.

DESCRIPTION OF THE INVENTION

The compounds object of the present invention are cyclic peptides ofgeneral formula (I):

wherein:

Xaa, independently of each other, is any amino acid;

Xbb is an alpha or beta amino acid containing at least three functionalgroups selected from the group consisting of:—COOH, —NH₂, —SH and —OH,

n is between 0 and 15, and

m is between 2 and 12.

Xbb is preferably selected from the group consisting of:

Lys, Asp, Glu, Cys, Orn, Dap, Dab, Gaba, epsilon-Aca and delta-Ava.

The invention also relates to compounds of formula (I) which arelabelled, either with the use of a chelating group or directly, withradioactive or paramagnetic metals or radioactive halogens and the saltsthereof with physiologically acceptable organic or inorganic bases orwith anions of physiologically acceptable organic or inorganic acids.

At least one of Xaa amino acids will preferably be a residue ofmethionine (Met), tyrosine (Tyr) or tyrosine-m-sulphonate (SO₃H-Tyr).

If the sulphate group is present on the tyrosine residue (Tyr orSO3H-Tyr), interaction with type A cholecystokinin receptor will beaided; conversely, a non-sulphated tyrosine residue (Tyr or SO3H-Tyr)sometimes may promote the interaction with type B cholecystokininreceptor.

The term “any amino acid” used above refers to the L and D isomers ofthe natural amino acids and “non-protein” amino acids commonly used inpeptide chemistry to prepare synthetic analogs of natural peptides, suchas alpha amino acids substituted and not substituted at the alpha andbeta positions of the L and D configurations, and unsaturated alpha/betaamino acids.

Examples of “non-proten” amino acids are norleucine, norvaline,alloisoleucine, allothreonine, homoarginine, thioproline,dehydroproline, hydroxyproline, pipecolic acid, azetidine acid,homoserine, cyclohexylglycine, alpha-amino-n-butyric acid,cyclohexylalanine, aminophenylbutyric acid, phenylalanine mono and di-substituted at the positions ortho, meta and para of the aromatic ring,O-alkylated derivatives of serine, threonine and tyrosine, S-alkylatedcysteine, epsilon-alkylated lysine, delta-alkylated ornithine, aromaticamino acids, substituted at the positions meta or para of the ring suchas phenylalanine-nitrate, -sulfate, -phosphate, -acetate, -carbonate,-methylsulfonate, -methylphosphonate, tyrosine-sulfate, -phosphate,-sulfonate, -phosphonate, para-amido-phenylalanine,C-alpha,alpha-dialkylated, amino acids such asalpha,alpha-dimethylglycine (Aib), alpha-aminocyclopropane-carboxylicacid (Ac3c), alpha-aminocyclobutane-carboxylic acid (Ac4c),alpha-aminocyclopentanecarboxylic acid (Ac5c),alpha-aminocyclohexanecarboxylic acid (Ac6c), diethylglycine (Deg),dipropylglycine (Dpg), diphenylglycine (Dph). Examples of beta-aminoacids are beta-alanine (beta-Ala), cis and trans 2,3-diaminopropionicacid (Dap).

Other non-protein amino acids are identified on the websitehttp://CHEMLIBRARY.BRI.NRC.CA/.

Preferred compounds of formula (I) are those wherein:

-   -   a) m is an integer between 4 and 8, n is an integer between 3        and 5, and the Xaa amino acids include at least one methionine        residue and one tyrosine residue in the sulphonated or        non-sulphonated form;    -   b) m is an integer between 4 and 8, n is an integer between 3        and 5, and the Xaa amino acids include at least one methionine        residue, one tyrosine residue in the sulphonated or        non-sulphonated form, and one lysine residue;    -   c) m is an integer between 4 and 8, n is an integer between 3        and 5, and the Xaa amino acids include at least one methionine        residue, one tyrosine residue in the sulphonated or        non-sulphonated form, one lysine residue or one amino acid        selected from ornithine, aspartic acid and glutamic acid.

Particularly preferred compounds are peptides of general formula (II):

wherein

m is 4;

n is an integer between 3 and 5;

Xaa1 and/or Xaa2 may be absent;

Xaa3 is Asp or Glu;

Xaa4 is Tyr or SO3H-Tyr;

Xaa10 is Phe or an amino acid selected from Leu, Ile, Val, Ala, Trp, Glyand Pro.

Even more particularly preferred are peptides of formula (III-VIII):

The compounds of the invention are synthetised by known techniques suchas solid-phase peptide synthesis, peptide synthesis in solution, organicchemistry synthesis methods, or any combination of those techniques.Synthesis methods based on appropriate combinations of solid-phasetechniques and conventional methods in solution, which involve lowproduction costs, especially on an industrial scale, will preferably beused. These methods involve solid-phase synthesis of the peptide,including branching with the use of protected amino acids withorthogonal functions, possibly solid-phase conjugation of themacrocycle, cleavage of the protective peptide from the resin, solutioncyclisation in diluted concentrations, and purification of the compound.

The compounds prepared according to general formula (I) can be labelledwith radioactive or paramagnetic metals or radioactive halogens, eitherdirectly or using a chelating group.

A further object of the present invention are the compounds of generalformula (IX):A-[Y]_(z)—C  (IX)

wherein

A is a peptide of general formula (I);

z is an integer between 0 and 5;

Y is a spacer chain respectively bonded to one of the functionalitiespresent on the side chains of the individual amino acids present inpeptide A, or to an N-terminal (—NH₂) group or a C-terminal (—CO₂H)group of A, and to C; when z is an integer between 2 and 5, units Y maybe the same or different from each other;

C is a chelating agent, bonded covalently to spacer chain Y or directlyto peptide A, or to more than one amino acid units of peptide A, whichis able to complex a paramagnetic metal or a radioisotopes.

Y is preferably a group of formula:

wherein

r, 1, and q are each independently 0 or 1, and p can vary between 0 and10, provided that at least one of 1, r and q is other than zero;

X is an O atom, or a —NR group wherein R is an H atom or an alkyl group(C₁-C₅);

K is a benzene nucleus, substituted or non-substituted, or a —CHR₁group, wherein R₁ is a hydrogen atom or a —COOH or —SO₃H group;

W is a —CO— or —CS— group.

Preferred compounds of formula (IX) are those in which the spacer chainsY have the following formulae (X), (XI) and (XII).

Particularly preferred are the compounds (IX) in which Y represents oneof the following groups:—(CH₂)₂—CO—, —(CH₂)₄—NH—, —(CH₂)₄—NH—CO—(CH₂)₂—CO—, —CH₂-Ph-CH₂—CO—,—CH₂-Ph-NH—CS—.

C preferably represents a chelating group selected from the groupconsisting of:

a residue of a polyaminopolycarboxylic acid and the derivatives thereof,in particular selected from diethylenetriaminopentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),[10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid (HPDO3A), 4-carboxy-5,8,11 -tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11 -triazatridecan-13-oic acid (BOPTA),N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethylglycine(EOB-DTPA),N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]-glycine(DTPA-BMA), 2-methyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (MCTA),(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraceticacid (DOTMA); or is the residue of a polyaminophosphate acid ligand orderivatives thereof, in particularN,N′-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N′-diacetic acid(DPDP) and ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP); oris the residue of a polyaminophosphonic acid ligand and derivativesthereof, or polyaminophosphinic acid and derivatives thereof, inparticular1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene(methylphosphonic)]acidand1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene-(methylphosphinic)]acid;or is the residue of macrocyclic chelants such as texaphrines,porphyrins, phthalocyanines; or isN,N-bis[2-[bis(carboxymethyl)amino]ethyl]L-glutamic acid (DTPA-GLU) orDTPA conjugated with Lys (DTPA-Lys).

The cyclic and/or branched peptide A can be linked to the chelatinggroup C, directly with a covalent bond between two functional groups ofA and C, or by the spacer chain Y and this can be obtained, for example,with the acid groups of the ligand, or by a suitable reactive grouppresent in the starting ligand, for example an amino group, or afunctional group present on a phenyl, etc.

Particularly preferred reactive groups present on C or Y, are selectedfrom the group consisting of —CO₂H, —NH₂, —NCS, —NHCSNHNH₂,—NHCSNH(CH₂)₂NH₂, —NCO, —NHNH₂, —NHCONHNH₂, —CHO.

Particularly preferred are the compounds of formula (IX) in which C is aresidue of the ligand DTPA, DO3A or DOTA.

Further examples of chelating groups C, which can be used in particularas chelating agents for radionuclides (such as Tc, Re, Cu, Ga) areamino/amide thiol derivatives that can be represented by the generalformula (XIII), wherein J is included within the range 0÷2 and hasgenerally unitary value.(N amino/amido)_(4−j)(S thiol derivative)_(j)  (XIII)

Preferred chelating groups of formula (XIII) include N₄ aminopropyleneoximes, diaminedioximes, hydrazines, N₃S triamide monothiols, N₂S₂diamido dithiols, diamine dithiols, monoamide monoaminedithiols andmonoamine monoamidedithiols.

A large number of applications of the chelating compounds of formula(XIII), labelled with Technetium or Rhenium are known in literature.

Preferred chelating agents C of metal ions and in particular of rheniumor technetium of formula (XIV a), (XIV b), (XIV c) and (XV) aredisclosed in EP 629,617, EP 544,412, U.S. Pat. No. 5,663,307 and U.S.Pat. No. 5,651,954, respectively, the content of which is incorporatedherein by reference, in particular as far as the definition of thegroups Q, R, R*, G1, G2 and R1 are concerned.

A further example of a compound which can be used as a chelating group Cof formula (XIII), is dimethylglycine-L-serine-L-cysteinylglycinamide(XVI), which has proved to be of considerable interest and suitable foruse as a bifunctionalised ligand for conjugation to peptides andproteins and for complexing of the radionuclide ^(99m)Tc or ¹⁸⁸Re. Otherderivatives which can be used as chelating agents C are disclosed inWO9933863, the content of which is incorporated herein by reference.

The functionalisation of the chelating groups of formula (XIII) enablesthem to be used as conjugates bonded with covalent bonds for a widerange of biologically active molecules.

In particular, in the preparation of compounds of formula (IX) used innuclear medicine diagnostics, chelating compounds C are selected in sucha way as to obtain corresponding stable complexes with radioisotopesthat emit gamma, beta or positron radiation, using for example theradioisotopes of Tc, Re, Tl, In, Cu, Ga, Rb or Y.

The use of ^(99m)Tc in nuclear medicine diagnostics is known, and itoffers a number of advantages which make it one of the most commonlyused radio nuclides. Its 6-hour half-life is short enough for theadministration of a dose of radiation that provides high-quality imageswithout risk to the patient's health.

Numerous techniques have also been developed which enable Tc to bebonded to various molecules with biological activity, such asantibodies, proteins and peptides.

The binding between the molecule and the radionuclide is usuallyobtained by one of the following methods:

-   a) direct binding of the radionuclide to the molecule concerned,    which is effected, for example, with the use of a reducing agent    that reduces the disulphide bridges of a peptide or a protein to two    hydrosulphide groups, which directly bind Tc(V);-   b) the use of a chelating agent C of the type described above,    generally bifunctionalised, in which one functionality is used for    the direct binding to the peptide (or to the compound, in accordance    with general formula Y, conjugated to A) and the other is used for    complexing with the radionuclide, which is performed before or after    binding with the biologically active molecule, as the case may be    (preformed-chelate or final-step-labelling method).

The radioactive complex is prepared by methods described in theliterature, such as by reaction of the functionalised chelating compoundwith a salt, in which Tc-99m pertechnetate is preferably used, in thepresence of a suitable reducing agent.

The reducing agents used include those reported in the literature, suchas dithionite, ferrous and stannous ions (e.g. tartrate, chloride andfluoride), or other solid-state reducing agents.

This type of complex is usually prepared with an inactive diagnostickit, previously prepared under aseptic conditions, which contains apredefined amount of the compound conjugated with the chelating agent inthe form of a freeze-dried powder, and of the reducing agent, bothformulated in the presence of suitable stabilising agents, surfactantsand/or buffers which can be used to prepare the pharmaceutical bulkproduct.

The solution containing the chelating agent is usually suitablyformulated and subsequently distributed into vials which arefreeze-dried and closed under nitrogen atmosphere to ensure that theproperties of the reducing agent (e.g. stannous chloride) present in thecomposition are maintained.

The inactive kits thus prepared are subsequently reconstituted, forexample with a sodium pertechnetate solution, to form the correspondingcomplexes with ^(99m)Tc, which are used in radiological diagnostics forfunctional and morphological examinations of organs of the human body,and in particular, for the compounds of the invention, which are usedfor imaging of tumors that over-express cholecystokinin receptors.

The use of radioactive rhenium isotopes as an alternative to technetiumisotopes has been proposed by Wong et al. (Wong, Inorganic Chemistry,vol. 36, 5799-5808, 1997), especially in the coordination state (V), andisotopes ¹⁸⁶Re and ¹⁸⁸Re have proved to be of particular interest innuclear medicine, having a large number of applications inradiopharmaceutical therapy.

Another class of chelating compounds C, which are suitable forconjugation with the compounds of the invention and can be used not onlyas chelators of paramagnetic metals (MRI) but also of radioactive metals(radiotherapy and radiodiagnostics), consists of polyazamacrocycles offormula (XVII). These chelating compounds contain at least one amine,thiol, carboxyl or carboxyl derivative group or a thiocarboxylate,present as free functionality and suitable for use in the conjugationreaction to spacer chain Y or peptide A, and according to the generalformula (IX).

For a complete description of the compounds of formula (XVII), see U.S.Pat. No. 6,093,382, which is incorporated herein by reference.

Compounds (I) and (IX) can form chelates with the bi-trivalent ions ofthe metal elements having atomic number ranging between 20 and 31, 39,42, 43, 44, 49, and between 57 and 83, with radioactive isotopes ofmetals or halogens (¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁷⁴Br, ⁷⁷Br and ⁸²Br)or paramagnetic metals (^(99m)Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³In,⁹⁰Yt, ⁹⁷Ru, ^(82m)Rb, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^(52m)Mn, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁵³Sm,¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁷⁷Lu, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi, ⁴⁷Sc, ¹⁴⁹Pm, ⁶⁷Cu, ¹¹¹Ag,¹⁹⁹Au, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁶¹Tb and ⁵¹Cr), possibly in the form of salts withphysiologically compatible bases or acids.

Particularly preferred are the complexes with Fe(²⁺), Fe(³⁺), Cu(²⁺),Cr(³⁺), Gd(³⁺), Eu(³⁺), Dy(³⁺), La(²⁺), Yb(³⁺) or Mn(²⁺) or withradioisotopes such as ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb,¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁴²Pr, ¹⁵⁹Gd and ²¹²Bi.

Preferred cations of inorganic bases suitable for salifying thecomplexes of the invention comprise, in particular, alkali oralkaline-earth metal ions such as potassium, sodium, calcium, magnesium.

Preferred cations of organic bases comprise those of primary, secondaryand tertiary amines, such as ethanolamine, diethanolamine, morpholine,glucamine, N-methylglucamine, N,N-dimethylglucamine.

Preferred anions of inorganic acids comprise, in particular, the ions ofhalo acids such as chlorides, bromides, iodides or other ions such assulfate.

Preferred anions of organic acids comprise those of acids conventionallyused in pharmaceutical technique for the salification of basicsubstances, such as acetate, succinate, citrate, fumarate, maleate,oxalate.

Preferred cations and anions of amino acids comprise, for example, thoseof taurine, glycine, lysine, arginine or omithine or of the aspartic andglutamic acids.

Compounds (I) and (IX) are also useful as cholecystokinin agonists orantagonists and, after labelling, either with the use of a chelatinggroup or directly with radioactive or paramagnetic metals or radioactivehalogens, as therapeutic and diagnostic agents to identify and locateprimary human tumors and their metastases which over-express type Aand/or B cholecystokinin receptors. Binding to the receptor may befollowed by a process of internalisation in the cells.

In particular, compounds of formula (IX), labelled with paramagneticmetals, have proved to be useful contrast media for magnetic resonance,especially for imaging of animal tumour cells which over-express type Aand/or type B cholecystokinin receptors.

Compounds (I) possess particular characteristics which make themsuitable for the purposes described above, in which:

-   a) they take on a structure in solution such that they can interact    specifically with type A and/or type B cholecystokinin receptors,    and have at least comparable affinity for CCK8;-   b) as a result of the presence of the cycle, they are particularly    stable to enzymatic degradation under physiological conditions;-   c) they take on a conformation such that the presence of a chelating    substituent does not interfere with binding to the receptor.

Compounds of formula (IX) can be prepared by conventional syntheticmethods. In particular, a compound of formula (IX) can be obtained withconvergent synthesis, which involves:

-   1) synthesis of a functionalised ligand, namely a ligand able to    coordinate a paramagnetic metal ion or the isotope of a radioactive    metal, which can also bind stably to the peptide, either directly or    through a suitable functional group;-   2) synthesis of the peptide;-   3) coupling reaction between the two different synthons, including    with the use of spacer unit Y;-   4) cleavage of any protective groups;-   5) complexing with a paramagnetic or radioactive metal ion.

The two synthons are conjugated by various known coupling methods widelyused in synthesis (see, for example, Brinkley, M., Bioconjugate Chem.1992, 3, 2), which involve for example the formation of an amide, athiourea or an ester.

Radioactive halogens used in therapy and diagnosis are known. Forexample, ¹²³I is known for its use in imaging, while ¹³¹I can be usednot only for imaging, but preferably in therapy. The bromineradionuclides ⁷⁵Br and ⁷⁶Br are used for diagnosis, while ⁷⁷Br is usedin radiotherapy. ¹⁸F and ²¹¹At are used in diagnosis and radiotherapy.

If the radionuclide is an isotope of a radioactive halogen, it can bebonded directly to peptide (I) by reacting with a Trp or Tyr residue.

The methods of labelling with iodine isotopes include not only directiodination with oxidative methods, but also the nucleophilicsubstitution reaction and the isotope exchange reaction. The choice oflabelling method in this case depends on the structure of the precursor,the problems associated with the purification techniques, and thecost-effectiveness of the process used.

An example of an indirect labelling method with radioactive halogenswhich can be used with compounds of formula (I) is described in U.S.Pat. No. 5,290,937, incorporated herein by reference, which enablesradio-labelled proteins of formula (XVIII) to be obtained:

wherein X is a radionuclide selected from ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁵Br, ⁷⁶Brand ⁷⁷Br, and A has the meaning described above for compounds of formula(I).

The use of the corresponding compounds labelled with radioisotopes whichemit gamma rays or alpha or beta particles enables a quantity of productsufficient to have a cytotoxic effect to be conveyed to the site inquestion. The radioactive decay of the isotope is generated at thetumour binding site, and determines the presence of a sufficientquantity of local ionising radiation to be toxic to the tumour cells.

The binding specificity for the cells that over-express cholecystokininreceptors enables these compounds to minimise the exposure of normalcells to the cytotoxic agents, thus providing an effective treatmentwith lower side effects.

In the case of compounds of formula (I) and (IX), both soluble and lesssoluble compounds are suitable for oral or parenteral administration.

Compounds for parental administration are preferably formulated as asterile aqueous suspension or solution, the pH of which can range, forexample, between 6.0 and 8.5.

These aqueous suspensions or solutions can be administered inconcentrations ranging between 0.002 and 1.0 molar.

These formulations can be freeze-dried in the form of powders andreconstituted at the time of use. For gastrointestinal use or injectioninto body cavities, these agents can be formulated as a suspension orsolution containing additives suitable to control their viscosity, forexample.

For oral administration they can be formulated in accordance withpreparation methods commonly used in pharmaceutical technology, possiblyas a coated formulation, in order to provide additional protectionagainst the acid pH of the stomach; this prevents release of thechelated metal ion, which particularly occurs at the pH values typicalof the gastric juices.

Other excipients, such as sweeteners and/or flavourings, can also beadded in accordance with known techniques.

Compounds of formula (I) and (IX), suitably formulated, are particularlyuseful for visualising pancreatic and oesophageal tumors and othertumors that over-express type A cholecystokinin receptors, and fortumors of the small cells of the lung, colon and gastrointestinal tract,medullary thyroid tumors, astrocytomas, ovarian stromal tumors and othertumors which over-express type B cholecystokinin receptors.

The experimental part describes the synthesis of the compound of formula(IV).

The compound of formula (IV) demonstrates a conformational behavioursimilar to that of the NMR complex between peptide CCK8 and receptorCCK-A.

FIG. 1 shows:

-   a) the structure of peptide CCK8 taken up into the complex with    N-terminal fragment 1-47 of the type A CCK receptor, as reported    by M. Pellegrini in Biochemistry, 38, 14775, 1999, and-   b) the expected structure of the cyclic compound of formula (IV).

The structure of the complex between non-sulphated peptide CCK8 and theN-terminal part of the type A cholecystokinin receptor, responsible forinteraction with the peptide hormone, is deposited in the Protein DataBank (http://www.rcsb.org) with the code “pdb1D6G.ent”. The N-terminalpart of the receptor (receptor filament) consists of 47 amino acidresidues, and represents the extracellular N-terminal arm and the firstpart of transmembrane helix 1 of the type A receptor.

The study of the structure of the complex (fragment 1-47 of type A CCKreceptor and peptide CCK8), performed with NMR spectroscopy, and thesubsequent binding study performed with fluorescence spectroscopy, werecarried out with peptide CCK8 in the non-sulphated form.

List of Abbreviations

For the nomenclature and abbreviations of the amino acids, referenceshould be made to the recommendations of the IUPAC-IUB Joint Commissionon Biochemical Nomenclature (Eur. J. Biochem. 1984, 138, p. 9); theamino acids are in the L configuration unless otherwise specified.

The other abbreviations used are:

Orn=ornithine, Nle, norleucine, Hyp=hydroxyproline,delta-Pro=dehydroproline, delta-Glu=alpha-beta-dehydroglutamic acid,alpha-Me-Glu=acid alpha-methyl-glutamic, Cha=cyclohexylalanine,at=alloisoleucine, Chg=cyclohexylglycine, Sar=sarcosine,Deg=diethylglycine, Dpg=dipropylglycine, Aib=alpha-aminoisobutyric acid,Dap=2,3 diaminopropionic acid, Dab=2,4 diaminobutyric acid,epsilon-Aca=epsilon-aminocaproic acid, delta-Ava=delta-aminovalericacid, beta-Ala=beta-alanine, Ac₃c=alpha-aminocyclopropanecarboxylicacid, Ac₄c=alpha-aminocyclobutanecarboxylic acid,Ac₅c=alpha-aminocyclopentanecarboxylic acid,Ac6c=alpha-aminocyclohexanecarboxylic acid,alpha-Ac₅c=alpha-aminocyclopentanecarboxylic acid,alpha-Ac₆c=alpha-aminocyclohexane-carboxylic acid,NO₂-Phe=nitrophenylalanine, SO₃H-Phe=sulfonated phenylalanine,PO₃H₂-Phe=phosphonated phenylalanine, PO₄H₂-Phe=phenylalanine phosphate,SO₄H-Phe=phenylalanine sulfate, SO₃H-Tyr=tyrosine-m-sulfonated,PO₃H₂-Tyr,=tyrosine-m-phosphonated, Boc=tert-butoxycarbonyl,Fmoc=fluorenylmethoxycarbonyl, TFA=trifluoroacetic acid,DCM=dichloromethane, DIEA=diisopropylethylamine, DMF=dimethylformamide,Obzl=benzyl ether, EDT=ethanedithiol, TIS=triisopropylsilane,HATU=O-(7-azabenzotriazol-yl)-1,1,3,3-tetramethyluronimtetrafluroborate, PyBop=benzotriazole-1-yl-oxi-tris-pyrrolidinium-phosphonium-hexafluophosphate, Trt=trityl,Pmc=2,2,5,7,8-pentamethyl-chroman-6-sulfonyl, Mtt=4-methyl-trityl,Dde=1-(4,4-dimethyl-2,6-dioxo-cyclohexyldene)-ethyl,Opfp=pentafluorophenyl ester, tBu=tert-butyl ether, OtBu=tert-butylester, Ac=acetyl, HPLC=high pressure liquid chromatography.

The following examples further illustrate the invention.

EXAMPLE 1

Synthesis of the cyclic peptide of formula (IV):

The peptide of formula (IV) contains the non-natural amino acid Dap(2,3-diaminopropionic acid), and this residue is used to form the cyclicpeptide thanks to the formation of the amide bond between the C-terminalcarboxyl group of the amino acid lysine and the amino group in position3 of 2,3-diaminopropionic acid.

The peptide was synthesised with the solid-phase peptide synthesisstrategy, using the Shimadzu automatic peptide synthesiser for batchsynthesis. In particular, the method that uses the Fmoc group asalpha-amino function protective group was employed. The solid mediumused for the synthesis was 2-chloro-trityl-chloride resin, whichconsists of a polystyrene medium with 2% divinylbenzene functionalisedwith the chloride group of 2-chloro-trityl; this resin allows thecarboxyl C-terminal peptide, completely protected on the side chains ofthe amino acids, to be obtained by hydrolysis in weakly acid conditions.

A synthesis scale of 0.176 mmoles was used. This synthesis scale wasobtained by using 400 mg of resin, which was previously partlyfunctionalised with the first amino acid, Fmoc-Lys(Boc).OH, inaccordance with the following procedure. A manual batch reactor wasused, and the amino acid deficit (0.6 equivalents) with respect to thefunctional groups present on the resin (0.83 mmoles/g) was added in DCM;an equivalent amount of PyBop and 4 DIEA equivalents, with respect tothe amount of amino acids used, were added. The number of mmoles ofamino acid bound to the resin was estimated on one aliquot by evaluatingthe amount of the Fmoc group present on the amino group in the alphaposition of the lysine residue and removed with a basic solutioncontaining 20% by volume of piperidine in DMF, and measuring theabsorbance of the chromophore effected in the UV region (lambda=290,epsilon₂₉₀=5.10³). After this procedure, the degree of substitution ofthe 2-chloro-trityl-Lys(Boc)-Fmoc resin was 0.44 mmoles/g. Synthesis ofthe peptide was then completed by transferring the resin bonded to thefirst amino acid (Fmoc-Lys(Boc)-) into the reactor of the synthesiser,and programming the synthesis with the following procedure: surplus ofindividual amino acids 2.5 equivalents, activating PyBop 1 equivalentwith respect to the amino acid, DIEA 1.5 equivalents with respect to theamino acid and PyBop, anhydrous DMF solvent, coupling time 60 minutes(single coupling). The amino acids used in sequence were as follows:Fmoc-Phe-OH, Fmoc-Asp(OtBu)—OH, Fmoc-Met-OH, Fmoc-Trp(Boc)—OH,Fmoc-Dap(Dde)—OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)—OH and Boc-Asp(OtBu)—OH.When assembly of the peptide on the resin had terminated, the Ddeprotective group of the side chain of the Dap residue was removed bytreating it with a 4% hydrazine solution in DMF. Two successive10-minute treatments were used for this deprotection reaction. Next, thecleavage reaction of the peptide partly protected by the resin wasperformed; the resin was placed on a porous septum. and treated with an0.5% solution (by volume) of TFA in dichloromethane for 3-5 minutes, theoperation being repeated 10 times. The acid washing solution wascollected in a flask containing 10% pyridine. After removing 90% of thestarting solvent volume by evaporation at reduced pressure, the peptidewas precipitated by addition of cold water and subsequently washed withwater. After checking with Maldi mass spectroscopy for the presence ofthe peptide with all the protective groups except for the one on the Dapside chain (molecular weight 1691), the cyclisation reaction wasperformed. The C-terminal carboxyl group on the lysine residue formed anamide bond with the amino group in position 3 of 2,3-diaminopropionicacid. The reaction was carried out in DMF at a peptide concentration of5.10⁻⁴M by adding the carboxyl function activator HATU in excess of 4equivalents and the DIEA base to a pH of 8.5. The mixture was kept understirring for 5 hours at 40° C. The solvent was then removed underreduced pressure, and the protective groups cleaved by the followingprocedure. The cyclic peptide was treated in a flask under stirring for2 hours with a mixture of TFA containing EDT (2.5%), TIS (1%) and water(2.5%). The homogeneity of the crude product was checked by analyticalHPLC; the crude product presented a main peak with a retention time of16.7 minutes (column: reverse phase C18; eluents: water with 1% TFA andacetonitrile with 1% TFA; elution gradient: water percentage from 80% to20% in 25 minutes). The product was purified by preparative HPLC, withthe same separation method as used on an analytical scale. The purity ofthe product obtained exceeded 98%, as confirmed by analytical HPLC, witha yield exceeding 50% of the expected theoretical yield. The identity ofthe product was confirmed with Maldi mass spectroscopy, which showed apeak at the expected mass value: 1202.

EXAMPLE 2

Determination of binding between compounds of formula (IV) and fragment1-47 of type A cholecystokinin receptor, CCK_(A)-R(1-47).

The binding between the compound prepared as described in example 1 andfragment 1-47 of type A cholecystokinin receptor was studied withfluorescence spectroscopy. Although the compound of formula (IV), as ithas no sulphate functions on the tyrosine residue, should have greateraffinity for type B cholecystokinin receptor, the experiment performedand the results obtained are entirely legitimate, because the receptorfragment used contains no residues (Arg 197) which are known to beresponsible for interaction with the sulphate group. The study of thestructure of the complex: fragment 1-47 of type A CCK receptor/peptideCCK8 carried out with NMR spectroscopy, and the subsequent binding studyperformed with fluorescence spectroscopy, used peptide CCK8 in thenon-sulphated form.

Fragment 1-47 of type A receptor, CCK_(A)-R(1-47), and the compound offormula (IV), both synthesised by solid-phase peptide synthesis methods,were used. Reference should be made to example 1 for the synthesis ofcompound (IV); for the synthesis of the receptor fragment, see R.Ragone, 47-53, 56, 2001, Biopolymers).

The interaction between receptor fragment CCKA-R(1-47) and compound (IV)was observed by monitoring the tryptophan fluorescence. The emissionspectra were recorded at room temperature using a Jasco FP-750spectrofluorimeter at the excitation wavelength of 296 nm to excite thetryptophan selectively. Small aliquots of a concentrated solution ofcompound (IV) dissolved in phosphate buffer at pH 7.2 were added to afixed volume of a solution of CCK_(A)-R(1-47) dissolved in the samesolvent, in the presence of a 10 mM solution of sodium dodecylsulphatemicelles, used to mimic the cell membrane. The final spectra used in thecalculations were corrected for dilution and the contributions of theindividual molecules isolated.

As shown in FIG. 2, evaluation of the graph gives the measurement of thebinding of the peptide of formula (IV) to fragment 1-47 of type Acholecystokinin receptor, CCKA-R(1-47), wherein the receptorconcentration is 2.6 μM in 10 mM of phosphate buffer, pH 7.2 (valuemeasured by absorbance. at 280 nm), in the presence of a 10 mM solutionof SDS micelles. Increasing quantities of compound of formula (IV) wereadded from a concentrated master solution containing 10 mM SDS micelles.The fluorescence was measured at 335 nm, and the binding curve resultingfrom the experiment agrees with a value of K_(d)=255 nM.

As will be seen in FIG. 2, which exemplifies a typical saturation curve,the fluorescence signal is gradually quenched as a result of thebinding. The experiments were performed at various receptorconcentrations, and fluorescence quenching was always observed. The sametitration curve is found for any given experiment, regardless of theemission wavelength chosen to perform the calculations. To distinguishthe modifications in fluorescence caused by the binding, thecontributions to fluorescence of the receptor and peptide isolated weresubtracted, and a small variation in fluorescence compared with thevalue measured was obtained. Although this involves a large statisticalvariation, it does not prevent correct evaluation of the quenching ofthe fluorescence. The fitting procedures used enabled us to estimate avalue of K_(d) in the 50-200 nM range, with a mean of 120 nM (standarddeviation ±27 nM). This value is in the same submicromolar affinityrange as found for non-sulphated linear binding of CCK8 to fragment 1-47of type A receptor or the entire type B receptor.

EXAMPLE 3

Evaluation of the biological activity of the peptide of formula (IV).

Biological tests have been carried out to confirm the activity of thecyclic compound of formula (IV), using neurone cells which express typeA cholecystokinin receptor. The activity of the compound of formula (IV)was compared with peptide CCK8 in the non-sulphated form on the tyrosineresidue. Both these compounds should show a lower level of activity thanthe corresponding analogs containing the sulphate group on the tyrosineresidue. The biological activity tests involved the use of myentericneurones as receptor-expressing cells, and the use of a confocalmicroscope with a suitable software package to measure peptide activity.The tests were based on the following assumptions:

-   1) myenteric neurones (of the intestinal wall) normally express type    A CCK receptor-   2) binding between CCK and the receptor induces excitation of the    cell (change in membrane potential), which can be recorded with    electrophysiology studies-   3) the cell excitation is coupled to the passage of calcium from the    outside to the inside of the cell through specific membrane channels-   4) if the calcium is rendered fluorescent (using calcium chelators    which modify the emission wavelength if they are excited at a    particular wavelength), the increased concentration of the    intracellular calcium in response to a stimulus can be recorded-   5) confocal laser microscopy with the aid of specific software    therefore enables these phenomena to be recorded, and the    fluorescence to be converted into the intracellular calcium    concentration.

When the neurones were exposed to non-sulphated peptide CCK8 and to thecyclic analog described in example 1 (perfusion for 60 seconds), it wasfound that both substances induce an increase in the intracellularcalcium concentration.

The test method is shown in FIG. 3. The graph, which shows two distinctcurves, relates to the same cell at two different times: before it wasinfused with non-sulphated peptide CCK8 and after washout (approx. 5mins.) with the cyclic peptide of formula (IV) described in example 1.The y-axis shows the fluorescence units, and the x-axis the perfusiontime (in seconds). The graph shows a first maximum peak (excitationafter perfusion with 75 mM K⁺), which is the stimulus used to identifythe neurones functionally, then the response to the CCK8 peptides andthe cyclic peptide of formula (IV), and finally 75 mM K⁺ again toconfirm cell viability.

Although the experiment does not evaluate the potency of action of thetwo molecules examined, the result indicates that the synthesised analogis able to bind to the specific receptor and induce a cell response.

EXAMPLE 4

Preparation of the functionalised cyclic peptide of formula (XIX).

The peptide component of compound (XIX) is the same as described inexample 1, and the side chain of the lysine residue present in thepeptide part of the compound of formula (IV), namelyN,N-bis[2-[bis(carboxymethyl)amino]ethyl]L-glutamic acid (DTPA-GLU), isalso used to bind the chelating agent covalently.

The compound of formula (XIX) was synthesised similarly to compound(IV), using the solid-phase peptide synthesis strategy and employingsynthesis procedures in manual reactors as well as the Shimadzuautomatic peptide synthesiser for batch synthesis. In particular, themethod that uses the Fmoc group as protective group of the alpha-aminofunction was employed. The solid medium used for the synthesis was2-chloro-trityl-chloride resin, which consists of a polystyrene mediumwith 2% divinylbenzene functionalised with the chloride group of2-chloro-trityl; this resin allows the carboxyl C-terminal peptide,completely protected on the side chains of the amino acids, to beobtained by hydrolysis in weakly acid conditions.

A synthesis scale of 0.180 mmoles was used. This synthesis scale wasobtained by using 400 mg of resin, which was previously partlyfunctionalised with the first amino acid, Dde-Lys(Fmoc)—OH, inaccordance with the following procedure. A manual batch reactor wasused, and the amino acid deficit (0.8 equivalents) with respect to thefunctional groups present on the resin (1.0.8 mmoles/g) was added inDCM; an equivalent amount of PyBop and 4 DIEA equivalents, with respectto the amount of amino acids used, were added. The number of mmoles ofamino acid bound to the resin was estimated on one aliquot by evaluatingthe amount of the Fmoc group present on the amino group in the epsilonposition of the lysine residue and removed with a basic solutioncontaining 20% by volume of piperidine in DMF, and measuring theabsorbance of the chromophore effected in the UV region (lambda=290,epsilon₂₉₀=5.10³). After this procedure, the degree of substitution ofthe 2-chloro-trityl-Lys(Boc)-Fmoc resin was 0.44 mmoles/g. The Fmocprotective group was then cleaved from the amino function in the epsilonposition of the Lys residue by treating it with a 20% solution ofpiperidine in DMF. Two successive 7-minute treatments were used for thedeprotection reaction. At a later stage we performed the condensationreaction of the chelating agent DTPA-GLU, protected on five of its sixcarboxyl functions with terbutyl groups, through its single freecarboxyl group, with the amino group in the epsilon position of the Lysresidue bonded to the resin. The reaction was performed using HATU asactivator, with a DTPA-GLU surplus of 2.5 equivalents, using the DIEAbase to stabilise the pH at 8.5. The Kaiser test was performed toconfirm that the coupling reaction was complete. The Dde protectivegroup present on the amino function in the alpha position of the Lysresidue was then cleaved by treating it with a 4% solution of hydrazinein DMF. Two successive 10-minute treatments were used for thisdeprotection reaction. This made it possible to perform the subsequentcoupling reaction of the amino acid Fmoc-Phe-OH, again with a manualsystem, under the same reaction conditions as described for thechelating agent DTPA-GLU. The Kaiser test again confirmed that thecoupling reaction was complete.

Synthesis of the peptide was then completed by transferring theLys(Glu-DTPA)-Phe-Fmoc resin to the reactor of the automatic synthesiserand programming synthesis with the following procedure: surplus of theindividual amino acids 2.5 equivalents, PyBop activator 1 equivalentwith respect to the amino acid, DIEA 1.5 equivalents with respect to theamino acid and PyBop, solvent anhydrous DMF, coupling time 60 minutes(single coupling). The amino acids used in sequence wereFmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH, Fmoc-Dap(Dde)-OH,Fmoc-Met-OH and Fmoc-Tyr(tBu)—OH and Boc-Asp(OtBu)—OH. When assembly ofthe peptide on the resin had terminated, the Dde protective group of theside chain of the Dap residue was removed by treating it with a 4%solution of hydrazine in DMF. Two successive 10-minute treatments wereused for this deprotection reaction. Next, the cleavage reaction of thepeptide partly protected by the resin was performed; the resin wasplaced on a porous septum and treated with an 0.5% solution in volume ofTFA in dichloromethane for 3-5 minutes, the operation being repeated 10times. The acid washing solution was collected in a flask containing 10%pyridine. After eliminating 90% of the starting solvent volume byevaporation at reduced pressure, the peptide was precipitated byaddition of cold water and subsequently washed with water. Afterchecking with Maldi mass spectroscopy for the presence of the peptidewith all the protective groups except for the one on the Dap side chain(molecular weight 2320), the cyclisation reaction was performed. TheC-terminal carboxyl group on the lysine residue formed an amide bondwith the amino group in position 3 of 2,3-diaminopropionic acid. Thereaction was carried out in DMF at a peptide concentration of 5.10⁻⁴ Mby adding the carboxyl function activator HATU in excess of 4equivalents and the DIEA base to a pH of 8.5. The mixture was kept understirring for 5 hours at 40° C. The solvent was then removed underreduced pressure, and the protective groups on the side chain of theamino acids and on the carboxyl functions of the DTPA chelating agentwere cleaved by the following procedure. The peptide compound wastreated in a flask under stirring for 2 hours with a mixture of TFAcontaining EDT (2.5%), TIS (1%) and water (2.5%). The homogeneity of thecrude product was checked by analytical HPLC; the crude productpresented a main peak with a retention time of 16.3 minutes (column:reverse phase C18; eluents: water with 1% TFA and acetonitrile with 1%TFA; elution gradient: water percentage from 80% to 20% in 25 minutes).The product was purified by preparative HPLC, with the same separationmethod as used on an analytical scale. The purity of the productobtained exceeded 98%, as confirmed by analytical HPLC, with a yieldexceeding 50% of the expected theoretical yield. The identity of theproduct was confirmed with Maldi mass spectroscopy, which showed thepeak at the expected mass value: 1650.

EXAMPLE 5

Preparation of the indium complex with formula (XX)

The compound of formula (XX) was synthesised by reacting the compoundhaving formula (XIX), prepared as described in example 4, with indiumtrichloride. The indium salt (II) was dissolved in a solution of 3%citrate buffer in water, and the resulting solution was added to asolution containing the compound of formula (XX), 0.5 indiumequivalents, in water at pH 7.8. After 24 hours the product was purifiedwith HPLC and characterised with Maldi mass spectroscopy, which showedthe peak at the expected mass value: 1762.

1. A compound having the general formula (IX):A-[Y]_(z)—C  (IX) wherein A is a peptide selected from:H-Xaa1-Xaa2-Xaa3-Xaa4-Met-Dap-Trp-Met-Asp-Xaal0-Lys

z is an integer between 0 and 5, Y is a spacer chain bonded to one ofthe functionalities present on the side chains of the individual aminoacids present in peptide A, or to an N-terminal (—NH₂) group or aC-terminal (—CO₂H) group of the peptide A, and to C; when z is aninteger between 2 and 5, Y may be the same or different from each other,and C is a chelating agent bonded covalently to spacer chain Y ordirectly to peptide A, or to a number of amino acid units of peptide A,which is able to complex a paramagnetic metal or a radioisotope, theircomplexes with radioactive or paramagnetic metals or radioactivehalogens, and salts thereof with physiologically acceptable organic orinorganic bases or with anions of physiologically acceptable organic orinorganic acids.
 2. A compound as claimed in claim 1, wherein C isselected from the group consisting of: LDTA; DTPA; LOB-DTPA; BOPTA;DTPA-BMA; DTPA-GLU; DTPA-Lys; DOTA; DOTMA; DO3A; HPDO3A; MCTA; DPDP;LDTP; 1,4,7,10-tetraazacyclododecano-1,4,7,10tetrakis[methylene(methylphosphonic)]acid;1,4,7,10-tetraazaciclododecano-1,4,7,10-tetrakis[methylene(methylphosphonic)]acid;texaphyrines, porphyrins, phthalocyanines; compounds of formula (Namino/amido)_(4−j) (S thiol)_(j) (XIII), wherein j has an integer valueof 0,1 or
 2. 3. A compound as claimed in claim 1, wherein Y is a groupof formula:

wherein r, 1, and q are each independently 0 or 1, and p can varybetween 0 and 10, provided that at least one of 1, r and q is other thanzero; X is an O atom, or a —NR group wherein R is an H atom or an alkylgroup (C₁-C₅); K is a benzene ring, substituted or non-substituted, or a—CHR₁ group wherein R₁ is a hydrogen atom or a —COOH or SO₃H group; andW is a —CO—or —CS—group.
 4. A compound as claimed in claim 3 whereinspacer chain—[Y]_(z)—has the following formula (X):

wherein z is an integer between 0 and 5; R is an H atom or an alkylgroup (C₁-C₅) and R₁ is a hydrogen atom or a —COOH or —SO₃H group; r, 1,and q are each independently 0 or 1, and p can vary between 0 and 10,provided that at least one of 1, r and q is other than zero.
 5. Acompound as claimed in claim 4 wherein the spacer chain —[Y]_(z)—has thefollowing formula (XI):

wherein z is an integer between 2 and 5; R is an H atom or an alkylgroup(C₁-C₅) and R₁ is a hydrogen atom or a —COOH or —SO₃H group; r, 1,and q are each independently 0 or 1, and p can vary between 0 and 10,provided that at least one of 1, r and q is other than zero.
 6. Acompound as claimed in claim 3, in which the spacer chain —[Y]_(z)—hasthe following formula (XII):

wherein z is an integer between 2 and 5; R is an H atom or an alkylgroup (C₁-C₅) and R₁ is a hydrogen atom or a —OOH or SO₃H group; W is a—CO—or a —CS—group; r, 1, and q are each independently 0 or 1, and p canvary between 0 and 10, provided that at least one of 1, r and q is otherthan zero.
 7. A compound as claimed in claim 1 wherein Y is one of thefollowing groups: —(CH₂)₂—CO—, —(CH₂)₄NH—, —(CH₂)₄—NH—CO—(CH₂)₂—CO—,—CH₂—Ph—CH₂—CO—, —CH₂—Ph—NH—CS—.
 8. A compound as claimed in claim 1,wherein C is DTPA, DTPA-Lys, or DTPA-GLU.
 9. A compound as claimed inclaim 1, wherein C is DOTA or DO3A.
 10. A compound as claimed in claim1, wherein said compound is a cholecystokinin agonist or antagonist. 11.A compound as claimed in claim 1 for the identification and localizationof primary animal and human tumors and their metastases whichover-express type A cholecystokinin receptors in higher quantities thanin non-pathological situations.
 12. A compound as claimed in claim 1 forthe identification and localization of primary animal and human tumorsand their metastases which over-express type B cholecystokinin receptorsin higher quantities than in non-pathological situations.
 13. A compoundas claimed in claim 1 for the identification and localization of primaryanimal and human tumors and their metastases which over-express type Aand/or B cholecystokinin receptors in higher quantities than innon-pathological situations.
 14. A pharmaceutical, or diagnosticcontrastographic or scintigraphic composition comprising a chelate asclaimed in claim 1 or a salt thereof.
 15. A diagnostic kit for thepreparation of a radiopharmaceutical composition wherein said kitcomprises an aseptically sealed vial containing a pre-determined amountof a compound as claimed in claim 1 and a sufficient quantity ofreducing agent used to label the compound with technetium-99m,rhenium-186 or rhenium-188.
 16. Diagnostic compositions for producingimage organs and/or tissues and/or cells of the human or animal body bynuclear resonance or scintigraphy comprising a complex of a compound ofclaim 1 with one or more radioactive or paramagnetic metals orradioactive halogens.
 17. A compound as claimed in claim 1 complexedwith a bi-trivalent ion of one or more metal elements having atomicnumbers ranging from 20 to 31, 39, 42-44, 49 or 57-83.
 18. A compound asclaimed in claim 1 complexed with one or more radioisotopes selectedfrom ¹²³I, ¹²⁵I, ¹³¹, ⁷⁴Br, ⁷⁶Br, ⁷⁵Br, ⁷⁷Br, ⁸²Br, ^(99m)Tc, ²⁰³Pb,⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³In, ⁹⁰Y, ⁹⁷Ru, ^(82m)Rb, ⁶²Cu, ⁶⁴Cu,^(52m)Fe, ⁵²Mn, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁷⁷Lu, ¹⁴²Pr, ¹⁵⁹Gd,²¹²Bi, ⁴⁷Sc, ¹⁴⁹Pm, ⁶⁷Cu, ¹¹¹Ag, ¹⁹⁹Au, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁶¹Tb and ⁵¹Cr.